Differentiation of a specific Trichoderma biological control agent by restriction fragment length polymorphism (RFLP) analysis

A single biological control isolate belonging to the species aggregate Trichoderma harzianum Rifai was differentiated from other closely related Trichoderma species and commonly isolated mycoflora by molecular assay. Restriction fragment length polymorphism (RFLP) analysis enabled the differentiation of the single isolate; a hybridising band of 1.1 kb being diagnostic. Cross-hybridisation occurred against the DNA from 12 Trichoderma isolates, representative of three species aggregates. The hybridisation probe used during the RFLP analysis was an amplification product produced during random amplification of polymorphic DNA (RAPD) polymerase chain reaction (PCR) analysis using DNA from T. harzianum isolate C65 as template. This amplification product was not visible following separation of amplification products generated during PCR using DNA from a test array of fungi as template. Diagnostic PCR, using primers designed from the sequence of the specific polymorphic amplification product failed to discriminate between C65 and other Trichoderma isolates, since products with equivalent size were generated. The ability to differentiate the isolate of T. harzianum from other closely related fungi will enable monitoring of the specific isolate in field trials to assess biological control ability.